Reagents for potentiometric magnesium ion selective electrode sensors and methods of production and use thereof

ABSTRACT

Reagents are disclosed for use with potentiometric magnesium ion selective electrodes, along with kits containing same as well as methods of use thereof.

CROSS REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCESTATEMENT

The subject application is a continuation of U.S. Ser. No. 16/059,095,filed Aug. 9, 2018; which is a continuation of U.S. Ser. No. 15/317,688,filed Dec. 9, 2016; which is a US national stage application filed under35 USC § 371 of International Application No. PCT/US2015/034668, filedJun. 8, 2015; which claims benefit under 35 USC § 119(e) of U.S.provisional Application No. 62/011,069, filed Jun. 12, 2014. The entirecontents of each of the above-referenced patent applications are herebyexpressly incorporated herein by reference.

BACKGROUND

The use of ion selective electrodes (ISEs) to determine the presence andquantity of various analytes in biological samples has become a usefuldiagnostic technique. Indeed, ISEs have been used to detect analytessuch as magnesium, sodium, potassium, calcium, and chloride, amongothers. Some of these ISEs are often housed within clinical diagnosticinstruments for simultaneous analysis of a large number of analytes.

One such use of the ISEs is for the determination of the amount ofmagnesium ions in a biological sample, specifically blood. Bloodcomprises many ions; the main ions present are magnesium ions (Mg²⁺),calcium ions (Ca²⁺), and sodium ions (Na⁺). For each type of ion, ISEshave a different response kinetic pattern, which causes the data to begreatly skewed if the ISEs are not calibrated to take into account thedifferent selectivities of the ions. Currently, the calibration ofpotentiometric ISEs for measuring ionized magnesium (“Mg ISE”) generallyencompasses calibrating the Mg ISE with three calibration reagents whichcharacterize the slope, intercept, and selectivity of the magnesium ionsagainst the calcium ions.

DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 illustrates the quality control recovery of a planar Mg ISEsensor that was calibrated with the three calibration reagents that havedecreased target amounts of Mg²⁺. AQC1 (diamonds), AQC2 (squares), andAQC3 (triangles) have target Mg²⁺ concentrations of 0.9 mmol/L, 0.6mmol/L, and 0.3 mmol/L, respectively. Each of the three calibrators hasa Ca²⁺:Mg²⁺ ratio of 1.14 to 1.34. As illustrated in FIG. 1, the planarMg ISE had an unstable recovery period for the first 3 to 5 days aftercoming in contact with each of these quality control reagents.

FIG. 2 illustrates the quality control recovery of a planar Mg ISEcalibrated with three calibration reagents constructed in accordancewith the presently disclosed and/or claimed inventive concept(s). Allthree calibration reagents have a Ca²⁺:Mg²⁺ ratio greater than 1.5. Likein FIG. 1, AQC1 (diamonds), AQC2 (squares), and AQC3 (triangles) havetarget Mg²⁺ concentrations of 0.9 mmol/L, 0.6 mmol/L, and 0.3 mmol/L,respectively. However, in this Example, AQC1 (diamonds), AQC2 (squares),and AQC3 (triangles) have Ca²⁺:Mg²⁺ ratios of 1.57, 1.92, and 2.89,respectively. As can be seen, the planar Mg ISE has a stable initialrecovery period that lasts throughout the lifespan of the magnesiumsensing membrane of the ISE.

FIG. 3 illustrates the variation in response kinetics for a planar MgISE in solutions with varying Ca²⁺:Mg²⁺ ratios. Normalized Delta mV=mV(t32)−mV (t0), which stands for mV difference between the end point at32 seconds and initial point at 0 seconds.

DETAILED DESCRIPTION

Before explaining at least one embodiment of the inventive concept(s) indetail by way of exemplary drawings, experimentation, results, andlaboratory procedures, it is to be understood that the inventiveconcept(s) is not limited in its application to the details ofconstruction and the arrangement of the components set forth in thefollowing description or illustrated in the drawings, experimentationand/or results. The inventive concept(s) is capable of other embodimentsor of being practiced or carried out in various ways. As such, thelanguage used herein is intended to be given the broadest possible scopeand meaning; and the embodiments are meant to be exemplary—notexhaustive. Also, it is to be understood that the phraseology andterminology employed herein is for the purpose of description and shouldnot be regarded as limiting.

Unless otherwise defined herein, scientific and technical terms used inconnection with the presently disclosed and/or claimed inventiveconcept(s) shall have the meanings that are commonly understood by thoseof ordinary skill in the art. Further, unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. Enzymatic reactions and purification techniquesare performed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. The nomenclatures utilized in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well-known and commonly used in the art.

All patents, published patent applications, and non-patent publicationsmentioned in the specification are indicative of the level of skill ofthose skilled in the art to which this presently disclosed and/orclaimed inventive concept(s) pertains. All patents, published patentapplications, and non-patent publications referenced in any portion ofthis application are herein expressly incorporated by reference in theirentirety to the same extent as if each individual patent or publicationwas specifically and individually indicated to be incorporated byreference.

All of the compositions and/or methods disclosed and/or claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this presentlydisclosed and/or claimed inventive concept(s) have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the compositions and/ormethods and in the steps or in the sequence of steps of the methoddescribed herein without departing from the concept, spirit and scope ofthe presently disclosed and/or claimed inventive concept(s). All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinventive concept(s) as defined by the appended claims.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The singular forms “a,” “an,” and “the”include plural referents unless the context clearly indicates otherwise.Thus, for example, reference to “a compound” may refer to 1 or more, 2or more, 3 or more, 4 or more, or greater numbers of compounds. The term“plurality” refers to “two or more.” The use of the term “or” in theclaims is used to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects. For example but not byway of limitation, when the term “about” is utilized, the designatedvalue may vary by ±20%, or ±10%, or ±5%, or ±1%, or ±0.1% from thespecified value, as such variations are appropriate to perform thedisclosed methods and as understood by persons having ordinary skill inthe art. The use of the term “at least one” will be understood toinclude one as well as any quantity more than one, including but notlimited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term “atleast one” may extend up to 100 or 1000 or more, depending on the termto which it is attached; in addition, the quantities of 100/1000 are notto be considered limiting, as higher limits may also producesatisfactory results. In addition, the use of the term “at least one ofX, Y and Z” will be understood to include X alone, Y alone, and Z alone,as well as any combination of X, Y and Z. The use of ordinal numberterminology (i.e., “first”, “second”, “third”, “fourth”, etc.) is solelyfor the purpose of differentiating between two or more items and is notmeant to imply any sequence or order or importance to one item overanother or any order of addition, for example.

As used in this specification and claim(s), the terms “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, the term “substantially” means that the subsequentlydescribed event or circumstance completely occurs or that thesubsequently described event or circumstance occurs to a great extent ordegree. For example, the term “substantially” means that thesubsequently described event or circumstance occurs at least 90% of thetime, or at least 95% of the time, or at least 98% of the time.

As used herein, the phrase “associated with” includes both directassociation of two moieties to one another as well as indirectassociation of two moieties to one another. Non-limiting examples ofassociations include covalent binding of one moiety to another moietyeither by a direct bond or through a spacer group, non-covalent bindingof one moiety to another moiety either directly or by means of specificbinding pair members bound to the moieties, incorporation of one moietyinto another moiety such as by dissolving one moiety in another moietyor by synthesis, and coating one moiety on another moiety.

The term “purified” as used herein means at least one order of magnitudeof purification is achieved compared to the starting material or of thenatural material, for example but not by way of limitation, two, three,four, or five orders of magnitude of purification of the startingmaterial or of the natural material. Thus, the term “purified” asutilized herein does not necessarily mean that the material is 100%purified, and therefore such term does not exclude the presence of othermaterial(s) present in the purified composition.

The term “sample” as used herein will be understood to include any typeof biological sample that may be utilized in accordance with thepresently disclosed and/or claimed inventive concept(s). Examples ofbiological samples that may be utilized include, but are not limited to,whole blood or any portion thereof (i.e., plasma or serum), saliva,sputum, cerebrospinal fluid (CSF), skin, interstitial fluid, tears,mucus, urine, swabs, combinations, and the like.

The term “wetup” as used herein will be understood to refer to thehydration process from the installation of a sensor in an analyzer to apoint at which a stable signal is obtained out of calibration reagents.

The term “recovery” as used herein, either alone or in connection withanother term (for example but without limitation, “quality controlrecovery,” “recovery period,” and “recovery elevation”), is understoodto mean the yield of an analytical process with comparison to anassigned value(s) or reference value(s).

Issues with the current methods of calibrating ISEs for measuringmagnesium ions stem from the calibration reagents that are currentlyused. These calibration reagents have been found to cause planar Mg ISEsensors to undergo a rapid wetup during the first few days aftercalibration, resulting in heightened response kinetics, and sometimestaking a week or more until the sensor is capable of providingreproducible measurements. Such a long wetup time significantly shortensthe useable lifespan of the sensor and prevents new and/or recentlycalibrated sensors from being readily useable.

It has previously been determined that the pattern of rapid wetup andthe resulting change in response kinetics for Mg ISEs is associated withthe ratio of calcium ions to magnesium ions in the calibration reagents.This is due to the Mg ISEs having different response kinetics forcalcium ions and magnesium ions. Specifically, the ionophores in Mg ISEshave previously been found to have an ionophore:Mg²⁺ andiononophore:Ca²⁺ stoichiometry of 1:1 and 2:1, respectively. See W.Zhang et al. (Analytical Sciences (2000) 16:11-18), which is herebyincorporated by reference in its entirety. This difference instoichiometry values suggests that the ionophores for Mg ISEs may have ahigher selectivity for calcium ions than magnesium ions and, therefore,the ratio of Ca²⁺ to Mg²⁺ should be calibrated to take into account thisselectivity.

It was reported that the ratio of Ca²⁺ to Mg²⁺ in solution affects theresponse kinetics of Mg selective electrodes (i.e., the time to steadystate). In general, the effect is produced by different rates ofdehydration of the magnesium and calcium ions, the latter rateapparently being higher [see, for example, Maj-Zurawska et al.(Analytica Chimica Acta (1990) 236:331-335); Mikhelson et al.(Electroanalysis (2001) 13:876-881); Marsoner et al. (Scand J Clin LabInvest (1994) 54(suppl 217):45-51); and Zhang et al. (AnalyticalSciences, incorporated supra)]. The kinetic discrimination of calciumand magnesium ions may be associated with the role of these ions in allprocesses at the membrane/solution interface, including thecalcium/magnesium biological pump or ion channel. During the initialwetup period, the Mg²⁺ ISE membrane/solution interface has a verydynamic dehydration process for the Mg²⁺ and Ca²⁺ ions, along withdifferent kinetics for each ion. When the Ca²⁺:Mg²⁺ ratio in solution isrelatively low, the net rate of dehydration for Ca²⁺ and Mg²⁺ at themembrane/solution interface may lead to unstable recovery evaluation ofaqueous samples (e.g., QC) and calibration response signals (see FIG.1), which could thus lead to significantly skewed measurements.

As described and/or claimed herein, it was presently discovered thatincreasing the amount of Ca²⁺ in the calibration reagents to a levelwherein the ratio of Ca²⁺:Mg²⁺ is similar to that of whole bloodminimizes the impact of the initial wetup period; the presentlydisclosed and/or claimed inventive concept(s) determined that, when thecalibration reagent has the same (or substantially similar) kineticpattern as the whole blood samples to be tested, the Mg ISE can beeffectively calibrated for measuring the magnesium ion content in blood.Additionally, applying the same Ca²⁺:Mg²⁺ ratio to the quality controlreagents and wash reagents likewise minimizes deviations in measurementstaken using the quality control reagents or after washing the magnesiumsensing membranes in the Mg ISEs.

It is to such reagents, as well as compositions, kits, and methodsrelated thereto, that the presently disclosed and/or claimed inventiveconcept(s) is directed.

Turning now to embodiments of the presently disclosed inventiveconcept(s), new and improved reagents are provided that can be used withMg sensors such that the Mg sensors exhibit increased stability andimproved response kinetics over prior art sensor/reagent combinations.The reagents of the presently disclosed and/or claimed inventiveconcept(s) can be used as calibration, quality control, and/or washreagents, and may be utilized with potentiometric ISEs for ionizedmagnesium, wherein the sensor/reagent combination exhibits improvedresponse kinetics and recovery stability during the initial period aftercalibrating the potentiometric ISEs for measuring ionized magnesium. Incertain embodiments, the new and improved reagents can be used, inparticular, with a solid-state planar magnesium sensing membrane for apotentiometric ion selective electrode that detects ionized magnesium ina biological sample.

In the presently disclosed and/or claimed inventive concept(s), thereagent is provided with a Ca²⁺ to Mg²⁺ distribution ratio that issubstantially similar to the ratio found in the biological sample to betested. In one embodiment, the biological sample is whole blood, whichhas a normal distribution ratio of Ca²⁺ to Mg²⁺ of around 2:1.Therefore, the reagent of this embodiment comprises calcium ions andmagnesium ions in a calcium:magnesium molar ratio in a range of fromabout 1.5 to about 3.25, such as but not limited to, a range of fromabout 1.7 to 3.25, or a range of from about 1.9 to about 2.1. In oneparticular embodiment, the reagent comprises calcium ions and magnesiumions in a calcium:magnesium molar ratio of 2:1.

In certain embodiments, the reagent may have a pH in a range of fromabout 6 to about 8, or from about 6.5 to about 7.8, or from about 6.8 toabout 7.2. In addition, the reagent may include additional components.For example but not by way of limitation, the reagent may furtherinclude one or more additional ions. Any other ion known in the art orotherwise contemplated herein may be present in the reagent and at anyconcentration, so long as the reagent and the potentiometric ionselective electrode can function in accordance with the presentlydisclosed and/or claimed inventive concept(s). For example but not byway of limitation, the reagent may further include sodium ions. In oneparticular embodiment, the reagent may further comprise at least 50mmol/L of sodium ions.

In other embodiments, the reagent may further comprise one or moresurfactants. Any surfactant(s) known in the art or otherwisecontemplated herein may be present in the reagent and at anyconcentration, so long as the reagent and the potentiometric ionselective electrode can function in accordance with the presentlydisclosed and/or claimed inventive concept(s). In certain embodiments,the surfactant present in the reagent may be a poly(ethylene oxide)surfactant, wherein the poly(ethylene oxide) surfactant may be utilizedat any concentration that allows the reagent and the potentiometric ionselective electrode to function in accordance with the presentlydisclosed and/or claimed inventive concept(s). A non-limiting example ofa poly(ethylene oxide) surfactant concentration that falls within thescope of the presently disclosed and/or claimed inventive concept(s) isless than about 100 mg/L.

Any poly(ethylene) surfactants known or otherwise contemplated withinthe art are capable of functioning as described herein and may beutilized in accordance with the presently disclosed and/or claimedinventive concept(s). Non-limiting examples of poly(ethylene oxide)surfactants that may be utilized in accordance with the presentlydisclosed and/or claimed inventive concept(s) are represented by thestructures of formulas I-III, as shown below.

In Formula I, n is in a range of from about 9 to about 10; in FormulaIII, n is about 100. One non-limiting example of a surfactantrepresented by the structure of Formula I (for example,t-octylphenoxypolyethoxyethanol) is sold under the trade name TRITON™X-100 (Sigma-Aldrich, St. Louis, Mo.). One non-limiting example of asurfactant represented by the structure of Formula II (for example,polyoxyethylene 23 lauryl ether) is known in the art by the productdesignation BRIJ® 35 (CAS No. 9002-92-0). A non-limiting example of asurfactant represented by the structure of Formula III (wherein n isabout 100) is polyoxyethylene(100) stearyl ether nonionic surfactant,which is known in the art by the product designation BRIJ® 700 (CAS No.9005-00-9). Particular non-limiting examples of the surfactantsrepresented by the structure of Formula III are disclosed in U.S. Pat.No. 8,496,900, issued to Zhang et al. on Jul. 30, 2013.

In one embodiment, the reagent is a quality control reagent, wherein thequality control reagent can be either an internal quality controlreagent or an external quality control reagent. As used herein, aninternal quality control reagent (also referred to as an “automaticquality control reagent”) is contained within the potentiometric ionselective electrode apparatus and comes into contact with the magnesiumsensing membrane at a pre-determined time and/or after a pre-determinedevent, wherein the pre-determined time/event may be mechanically orelectronically programed into the potentiometric ion selectiveelectrode. Additionally, as used herein, an external quality controlreagent is a quality control reagent that is initially separate (i.e.,external) from the potentiometric ion selective electrode apparatus andis made to come into contact with the magnesium sensing membrane at apre-determined time and/or after a pre-determined event by actions of anoperator of the potentiometric ion selective electrode apparatus.

Another embodiment of the presently disclosed and/or claimed inventiveconcept(s) is directed to a method of calibrating a magnesium sensingmembrane for a potentiometric ion selective electrode. In the method,the magnesium sensing membrane is contacted with one or more of thecalibration reagents described or otherwise contemplated herein above.In one particular embodiment, the presently disclosed and/or claimedinventive concept(s) is directed to a method of calibrating asolid-state planar magnesium sensing membrane for a potentiometric ionselective electrode, wherein a solid-state planar magnesium sensingmembrane is contacted with one or more calibration reagents(s) describedor otherwise contemplated herein above. In one particular embodiment,the method of calibrating the magnesium sensing membrane for apotentiometric ion selective electrode comprises contacting themagnesium sensing membrane with at least three separate calibrationreagents described or otherwise contemplated herein above tocharacterize the slope, intercept, and selectivity against calcium ions.

Another embodiment of the presently disclosed and/or claimed inventiveconcept(s) is directed to a method of monitoring the quality control ofa magnesium sensing membrane for a potentiometric ion selectiveelectrode. In the method, the magnesium sensing membrane is contactedwith one or more of the reagents described or otherwise contemplatedherein above. In one particular embodiment, the presently disclosedand/or claimed inventive concept(s) is directed to a method ofmonitoring the quality control of a solid-state planar magnesium sensingmembrane for a potentiometric ion selective electrode. In the method, asolid-state planar magnesium sensing membrane is contacted with one ormore of the reagents described or otherwise contemplated herein above,wherein at least one reagent is a quality control reagent. In the methodof monitoring the quality control of a magnesium sensing membrane for apotentiometric ion selective electrode, the magnesium sensing membranemay be contacted with at least three separate quality control reagentsdescribed or otherwise contemplated herein above to characterize theslope, intercept, and selectivity against calcium ions.

Another embodiment of the presently disclosed and/or claimed inventiveconcept(s) is directed to a method of washing a magnesium sensingmembrane for a potentiometric ion selective electrode. In the method,the magnesium sensing membrane is contacted with one or more of the washreagents described or otherwise contemplated herein above. Inparticular, the presently disclosed and/or claimed inventive concept(s)is directed to a method of washing a solid-state planar magnesiumsensing membrane for a potentiometric ion selective electrode; in themethod, a solid-state planar magnesium sensing membrane is contactedwith one or more of the wash reagents described or otherwisecontemplated herein above.

While certain embodiments of the reagents described and contemplatedherein may be disclosed as being a “calibration reagent,” a “qualitycontrol reagent,” or a “wash reagent,” it is to be understood that thesedesignations are not to be perceived as limiting; a single formulationof reagent may possess two or three of the calibration, quality control,and/or wash functions. Therefore, while the above methods are disclosedas requiring a “calibration reagent,” a “quality control reagent,” or a“wash reagent,” this requirement is simply based upon the function ofthe reagent and not on the composition thereof. A single reagent may becapable as functioning as two or more of a calibration reagent, aquality control reagent, and a wash reagent. Thus, it will be understoodthat the methods of (i) calibrating, (ii) monitoring the quality controlof, and (iii) washing a magnesium sensing membrane may all utilize asingle reagent composition that is capable of providing all threefunctions.

Yet other embodiments of the presently disclosed and/or claimedinventive concept(s) are directed to kits that include one or more ofany of the reagents described or otherwise contemplated herein. When twoor more reagents are present, the combination of reagents may be of thesame function and/or different function. In one embodiment, the kit mayinclude at least one calibration reagent, at least one quality controlreagent, at least one wash reagent, or any combination thereof. Forexample but not by way of limitation, the kit may include at least onecalibration reagent, at least one quality control reagent, and at leastone wash reagent. In other embodiments, the kit may include combinationsof reagents that possess the same function. For example but not by wayof limitation, the kit may include at least three calibration reagents;in addition, this kit may further include at least three quality controlreagents and/or at least one wash reagent.

The kits of the presently disclosed and/or claimed inventive concept(s),which include one or more of any of the reagents or combinations ofreagents described or otherwise contemplated herein, may further includea solid-state planar magnesium PVC (polyvinyl chloride) sensing membranefor a potentiometric magnesium ion selective electrode. Any solid-stateplanar magnesium PVC sensing membrane known in the art or otherwisecontemplated herein may be utilized in accordance with the presentlydisclosed and/or claimed inventive concept(s). Non-limiting examples ofmembranes that may be utilized include, but are not limited to, membraneformulations containing Mg²⁺ ionophores of malondiamide derivatives, aswell as those disclosed in Zhang et al. (Analytical Sciences,incorporated supra) and Lim et al. (Pure Appl. Chem. (2004) 75:753-764).Non-limiting examples of specific ionophores that may be utilizedinclude, but are not limited to, Tris(malondiamides) derivatives (suchas but not limited to, ETH5506, ETH5504, ETH7025, ETH3832, K22B5, andthe like), as well as those disclosed in Zhang et al. (AnalyticalSciences, incorporated supra). Other non-limiting examples of specificmembranes, ionophores, and lipophilic salts that may be utilized includethose disclosed in Maj-Zurawska et al., Mikhelson et al., and Marsoneret al. (all incorporated supra). The entire contents of each of theabove references are hereby expressly incorporated herein.

Another non-limiting example of a magnesium sensing membrane for apotentiometric ion selective electrode that may be utilized inaccordance with the presently disclosed and/or claimed inventiveconcept(s) is disclosed in co-pending Application U.S. Ser. No.61/981,277, filed Apr. 18, 2014, which is hereby expressly incorporatedherein in its entirety.

In certain embodiments, the kits of the presently disclosed and/orclaimed inventive concept(s), which include one or more of any of thereagents or combinations of reagents described or otherwise contemplatedherein, may further include a potentiometric magnesium ion selectiveelectrode comprising the solid-state planar magnesium membrane asdescribed herein above.

In addition, the kits may further contain other reagent(s) forconducting any of the particular methods described or otherwisecontemplated herein. The nature of these additional reagent(s) willdepend upon the particular assay format, and identification thereof iswell within the skill of one of ordinary skill in the art.

The components/reagents may each be disposed in separatecontainers/compartments of the kit, or various components/reagents canbe combined in one or more containers/compartments of the kit, dependingon the competitive nature of the components/reagents and/or thestability of the components/reagents. The kit can further include otherseparately packaged reagents for conducting an assay. The relativeamounts of the various components/reagents in the kits can vary widelyto provide for concentrations of the components/reagents thatsubstantially optimize the reactions that need to occur during the assaymethods and further to optimize substantially the stability/sensitivityof an assay. Positive and/or negative controls may be included with thekit. The kit can further include a set of written instructionsexplaining how to use the kit. For example, but not by way oflimitation, the kit may further include instructions for using thereagents to rinse, calibrate, operate, and/or monitor quality control ofthe potentiometric ion selective electrode. A kit of this nature can beused in any of the methods described or otherwise contemplated herein.

EXAMPLE

An Example is provided hereinbelow. However, the presently disclosedand/or claimed inventive concept(s) is to be understood to not belimited in its application to the specific experimentation, results, andlaboratory procedures disclosed herein below. Rather, the Example issimply provided as one of various embodiments and is meant to beexemplary, not exhaustive.

In this Example, the instability of QC (AQC) recovery of apotentiometric magnesium ion selective electrode was studied over auselife of four weeks. As shown herein below, reformulation of thecalibration reagents utilized with the ISE to provide the reagents toprovide the desired Ca²⁺:Mg²⁺ ratio improved the performance stabilityof the Mg sensor.

FIG. 1 shows an AQC recovery plot of the response data obtained using apotentiometric ISE with a solid-state planar magnesium sensing membranethat was calibrated with calibration reagents of the prior art. One ofthree prior art calibration reagents had a Ca²⁺:Mg²⁺ ratio of about1.34. Three aqueous QC samples were tested at three different targetMg²⁺ concentrations (AQC1: 0.9 mmol/L; AQC2: 0.6 mmol/L; and AQC3: 0.3mmol/L). The sensor had blood contact since installation.

As can be seen, unstable recovery plots were observed at all threelevels of AQCs tested, especially during the first week of uselife. The“humps” observed over the first 5-10 days of uselife indicate that theMg²⁺ concentrations of the reagents were incorrectly detected at a levelas much as 2×-3× above their actual concentration during this time. Thisobservation of “hump” QC recovery is explained by slow dynamiccomplexation between the magnesium ion ionophore in the sensor membraneand the Ca²⁺ and Mg²⁺ ions in the calibration reagents. Thestoichiometry number between ionophore:Mg²⁺ is believed to be 1:1, andthe stoichiometry number between ionophore and Ca²⁺ is believed to be2:1 (see Zhang et al., Analytical Sciences, incorporated supra).

In contrast, FIG. 2 illustrates an AQC recovery plot of the responsedata obtained using a potentiometric ISE with a solid-state planarmagnesium sensing membrane that was calibrated with calibration reagentsof the presently disclosed and/or claimed inventive concept(s) thatpossessed a Ca²⁺:Mg²⁺ ratio above the prior art level of <1.5. Thesecalibration reagents had Ca²⁺:Mg²⁺ ratios in a range of from about 1.7to about 3.25 (i.e., 1.57 for AQC1, 1.92 for AQC2, and 2.89 for AQC3),and the reagents were tested at the same three target Mg²⁺concentrations (AQC1: 0.9 mmol/L; AQC2: 0.6 mmol/L; and AQC3: 0.3mmol/L). As above, the sensor had blood contact since installation.

Unlike the prior art reagents, the substantially linear lines of FIG. 2indicate stable recovery plots over the entire uselife window for allthree levels of AQCs tested. These stable response data measurements forthe useable life of the magnesium sensor were observed when the ratio ofCa²⁺:Mg²⁺ is increased to be above 1.5 and closer to (or above) 2, whichis the approximate Ca²⁺: Mg²⁺ ratio for whole blood samples. It isbelieved that a calibration reagent setting of a Ca²⁺:Mg²⁺ ratio in arange of from about 1.7 to about 3.25 helps build up dynamic equilibriabetween ionophore in the solid state sensor and the Ca²⁺ and Mg²⁺ ionsin the calibration reagents.

It has been determined herein that, if the ratio of Ca²⁺:Mg²⁺ is too farbelow 2:1 (i.e., 1.5:1 or lower) during the initial contact periodbetween the sensor membrane and the calibration reagents, it leads to aslow procedure to reach complexation equilibrium between the ionophoreand the Mg²⁺ and Ca²⁺ ions in the magnesium sensing membrane of the ISE(i.e., at the membrane interface and in the membrane bulk), as seen inFIG. 1 and described herein above. When the sensor contacts a bloodsample during the initial period, the adsorbed protein layer functionsas an ion-exchanger that responds to electrolytes following the normaldistribution ratio in blood for Ca²⁺ and Mg²⁺ (Ca²⁺=1.2 mmol/L toMg²⁺=0.5 mmol/L). As such, the presently disclosed and/or claimedinventive concept(s) have determined that a ratio of Ca²⁺:Mg²⁺ inreagents should be close to 2:1 so that the sensor has identicalperformance in all reagents (calibration reagents, Wash, AQCs, etc.).

In a calibration setting containing two to three calibration reagents,if one reagent has a Ca²⁺:Mg²⁺ ratio that is well below 2:1 (e.g.,<1.5), the thermodynamic complexation process between ionophore and Mg²⁺and Ca²⁺ ions will be sluggish (FIG. 3). It will take a long time tobuildup a steady-state membrane potential response in all reagents whenthey possess different Ca²⁺:Mg²⁺ ratios (membrane potential=phaseboundary potential+membrane diffusion potential), ultimately leading toan unstable initial response period. Adjusting the Ca²⁺:Mg²⁺ ratio inall of the reagents (i.e., all calibration, wash, and QC reagentsutilized with a particular ISE) to a ratio in the range of from about1.5 to about 3.25 will accelerate such thermodynamic process. Thus, theMg²⁺ sensor can reach a steady-state potential response in all reagents.

Additionally, ionic strength in all of the reagents should be adequatelyhigh enough (i.e., an ionic strength between at least 50-160 mmol/L) sothat the concentration values of Mg²⁺ and Ca²⁺ are not significantlyaffected by activity coefficient differences among the reagents. Asshown in Table 1, ionic strength (IS) has a significant impact on themolal activity variation of divalent cations. If one of the calibrationreagents has an IS of 50 mmol/L (Na⁺) and the other calibrationreagent(s) has an IS of 125 mmol/L, the molal activity variation of thedivalent cation cannot be neglected. For Mg²⁺ and Ca²⁺ concentrations of0.5 mmol/L and 1.2 mmol/L, respectively, the molal activities of thecations are 0.21 mmol/L (Mg²⁺) and 0.44 mmol/L (Ca²⁺) in 125 mmol/L Na⁺solution. However, in 25 mmol/L Na⁺ solution, the molal activities ofthe cations become 0.29 mmol/L (Mg²⁺) and 0.66 mmol/L (Ca²⁺). Since apotentiometric sensor responds only to the molal activity (as opposed tothe mass concentration), molal activity variation induced by ionicstrength can lead to biased calibration results (slope, selectivity, andintercept). With such biased calibration parameters, blood samplerecovery can be wrongly calculated.

TABLE 1 Impact of Ionic Strength on Activity Coefficient and MolalActivity of Cations in Aqueous Solution Ionic Strength K⁺ Ca²⁺ Mg²⁺(Na⁺, mmol/L) (4 mmol/L) (1.2 mmol/L) (0.5 mmol/L) Activity 150 0.7130.348 0.398 coefficient 125 0.729 0.367 0.414 100 0.747 0.391 0.436 750.770 0.424 0.464 50 0.800 0.471 0.505 25 0.844 0.553 0.578 10 0.8890.652 0.667 Molal activity 150 2.85 0.42 0.20 (mmol/L) 125 2.92 0.440.21 100 2.99 0.47 0.22 75 3.08 0.51 0.23 50 3.20 0.57 0.25 25 3.38 0.660.29 10 3.56 0.78 0.33

Therefore, in accordance with the presently disclosed and/or claimedinventive concept(s), there have been provided reagents, as well as kitscontaining same and methods of use thereof, that fully satisfy theobjectives and advantages set forth hereinabove. Although the presentlydisclosed and/or claimed inventive concept(s) has been described inconjunction with the specific drawings, experimentation, results, andlanguage set forth herein above, it is evident that many alternatives,modifications, and variations will be apparent to those of ordinaryskill in the art. Accordingly, it is intended to embrace all suchalternatives, modifications, and variations that fall within the spiritand broad scope of the presently disclosed and/or claimed inventiveconcept(s).

The following is a numbered list of non-limiting, illustrativeembodiments of the inventive concepts disclosed herein:

1. A reagent for a solid-state planar magnesium sensing membrane for apotentiometric ion selective electrode that detects ionized magnesium ina biological sample, the reagent comprising calcium ions and magnesiumions present in a calcium:magnesium molar ratio in a range of from about1.5:1 to about 3.25:1.

2. The reagent of illustrative embodiment 1, wherein thecalcium:magnesium molar ratio is in a range of from about 1.7:1 to about3.25:1.

3. The reagent of illustrative embodiment 1, wherein thecalcium:magnesium molar ratio is about 2:1.

4. The reagent of any of illustrative embodiments 1 to 3, wherein thereagent has a pH in a range of from about 6 to about 8.

5. The reagent of any of illustrative embodiments 1 to 3, wherein thereagent has a pH in a range of from about 6.5 to about 7.8.

6. The reagent of any of illustrative embodiments 1 to 5, furthercomprising sodium ions at a concentration of at least 50 mmol/L.

7. The reagent of any of illustrative embodiments 1 to 6, furthercomprising a surfactant.

8. The reagent of illustrative embodiment 7, wherein the surfactantcomprises a poly(ethylene oxide) surfactant.

9. The reagent of illustrative embodiment 8, wherein the poly(ethyleneoxide) surfactant is represented by the structure of formula I:

wherein n is in the range of from about 9 to about 10.

10. The reagent of illustrative embodiment 8, wherein the poly(ethyleneoxide) surfactant is represented by the structure of formula II:

HO—(CH₂—CH₂—O—)₂₃—C₁₂H₂₅  Formula II

11. The reagent of illustrative embodiment 8, wherein the poly(ethyleneoxide) surfactant is represented by the structure of formula III:

wherein n is 100.

12. The reagent of illustrative embodiment 8, wherein the concentrationof the poly(ethylene oxide) surfactant is less than about 100 mg/L.

13. The reagent of any of illustrative embodiments 1 to 12, wherein thereagent is a calibration reagent.

14. The reagent of any of illustrative embodiments 1 to 12, wherein thereagent is a quality control reagent.

15. The kit of illustrative embodiment 14, wherein the quality controlreagent is an external quality control reagent.

16. The kit of illustrative embodiment 14, wherein the quality controlreagent is an internal quality control reagent.

17. The reagent of any of illustrative embodiments 1 to 12, wherein thereagent is a wash reagent.

18. A method of calibrating a solid-state planar magnesium sensingmembrane for a potentiometric ion selective electrode comprising thestep of contacting a solid-state planar magnesium sensing membrane withthe calibration reagent of illustrative embodiment 13.

19. A method of monitoring the quality control of a solid-state planarmagnesium sensing membrane for a potentiometric ion selective electrodecomprising the step of contacting a solid-state planar magnesium sensingmembrane with the quality control reagent of illustrative embodiment 14.

20. A method of washing a magnesium sensing membrane for apotentiometric ion selective electrode comprising the step of contactinga solid-state planar magnesium sensing membrane with the wash reagent ofillustrative embodiment 17.

21. A kit comprising: at least one calibration reagent of illustrativeembodiment 13.

22. The kit of illustrative embodiment 21, wherein the at least onecalibration reagent is further defined as at least two calibrationreagents, and wherein each calibration reagent is a calibration reagentof illustrative embodiment 13.

23. The kit of illustrative embodiment 21, further comprising: at leastone quality control reagent of illustrative embodiment 14; and/or atleast one wash reagent of illustrative embodiment 17.

24. The kit of illustrative embodiment 19, wherein the at least onecalibration reagent is further defined as at least three calibrationreagents, and wherein each calibration reagent is a calibration reagentof illustrative embodiment 13, and wherein the kit further comprises: atleast three quality control reagents of illustrative embodiment 14; andat least one wash reagent of illustrative embodiment 17.

25. A kit comprising: at least one calibration reagent of illustrativeembodiment 13; and a potentiometric ion selective electrode comprising asolid-state planar magnesium sensing membrane.

26. The kit of illustrative embodiment 25, wherein the at least onecalibration reagent is further defined as at least two calibrationreagents, and wherein each calibration reagent is a calibration reagentof illustrative embodiment 13.

27. The kit of illustrative embodiment 25, further comprising: at leastone quality control reagent of illustrative embodiment 14; and/or atleast one wash reagent of illustrative embodiment 17.

28. The kit of illustrative embodiment 25, wherein the at least onecalibration reagent is further defined as at least three calibrationreagents, and wherein each calibration reagent is a calibration reagentof illustrative embodiment 13, and wherein the kit further comprises: atleast three quality control reagents of illustrative embodiment 14; andat least one wash reagent of illustrative embodiment 17.

What is claimed is:
 1. A method of calibrating a solid-state planarmagnesium sensing membrane for a potentiometric ion selective electrodethat detects ionized magnesium in a biological sample, the methodcomprising: calibrating the magnesium sensing membrane with at leastthree separate calibration reagents, wherein each of the calibrationreagents comprises calcium ions and magnesium ions present in acalcium:magnesium molar ratio in a range of from about 1.5:1 to about3.25:1.
 2. The method of claim 1, wherein the calcium:magnesium molarratio in each of the reagents is in a range of from about 1.7:1 to about3.25:1.
 3. The method of claim 1, wherein the calcium:magnesium molarratio in each of the reagents is about 2:1.
 4. The method of claim 1,wherein each of the calibration reagents has a pH in a range of fromabout 6 to about
 8. 5. The method of claim 1, wherein at least one ofthe calibration reagents has a pH in a range of from about 6.5 to about7.8.
 6. The method of claim 1, wherein each of the calibration reagentsfurther comprises sodium ions at a concentration of at least 50 mmol/L.7. The method of claim 1, wherein each of the calibration reagentsfurther comprises a surfactant.
 8. The method of claim 7, wherein thesurfactant present in each calibration reagent comprises a poly(ethyleneoxide) surfactant.
 9. The method of claim 8, wherein the poly(ethyleneoxide) surfactant is represented by the structure of formula I:

wherein n is in the range of from about 9 to about
 10. 10. The method ofclaim 8, wherein the poly(ethylene oxide) surfactant is represented bythe structure of formula II:HO—(CH₂—CH₂—O—)₂₃—C₁₂H₂₅  Formula II
 11. The method of claim 8, whereinthe poly(ethylene oxide) surfactant is represented by the structure offormula III:

wherein n is
 100. 12. The method of claim 8, wherein the concentrationof the poly(ethylene oxide) surfactant present in each calibrationreagent is less than about 100 mg/L.
 13. The method of claim 1, furthercomprising the step of contacting the magnesium sensing membrane with atleast one additional reagent selected from a wash reagent and a qualitycontrol reagent, wherein the at least one additional reagent comprisescalcium ions and magnesium ions present in a calcium:magnesium molarratio in a range of from about 1.5:1 to about 3.25:1.
 14. The method ofclaim 1, wherein the magnesium sensing membrane is subjected to multiplecalibrations with the at least three separate calibration reagents andis stable for at least four weeks.
 15. A method of calibrating asolid-state planar magnesium sensing membrane for a potentiometric ionselective electrode that detects ionized magnesium in a biologicalsample, the method comprising: calibrating the magnesium sensingmembrane with at least three separate calibration reagents, wherein eachof the calibration reagents comprises calcium ions and magnesium ionspresent in a calcium:magnesium molar ratio in a range of from about1.5:1 to about 3.25:1, and wherein each of the calibration reagentsfurther comprises a poly(ethylene oxide) surfactant selected from thegroup consisting of: (a) the poly(ethylene oxide) surfactant isrepresented by the structure of formula I:

wherein n is in the range of from about 9 to about 10; (b) thepoly(ethylene oxide) surfactant is represented by the structure offormula II:HO—(CH₂—CH₂—O—)₂₃—C₁₂H₂₅  Formula II; and (c) the poly(ethylene oxide)surfactant is represented by the structure of formula III:

wherein n is
 100. 16. The method of claim 15, wherein each one of thereagents has a pH in a range of from about 6 to about 8, and whereineach of the reagents further comprises sodium ions at a concentration ofat least 50 mmol/L.
 17. The method of claim 15, further comprising thestep of contacting the magnesium sensing membrane with at least oneadditional reagent selected from a wash reagent and a quality controlreagent, wherein the at least one additional reagent comprises calciumions and magnesium ions present in a calcium:magnesium molar ratio in arange of from about 1.5:1 to about 3.25:1.
 18. The method of claim 15,wherein the magnesium sensing membrane is subjected to multiplecalibrations with the at least three separate calibration reagents andis stable for at least four weeks.
 19. A method of calibrating asolid-state planar magnesium sensing membrane for a potentiometric ionselective electrode that detects ionized magnesium in a biologicalsample, the method comprising: calibrating the magnesium sensingmembrane with at least three separate calibration reagents, wherein eachof the calibration reagents comprises: calcium ions and magnesium ionspresent in a calcium:magnesium molar ratio in a range of from about1.5:1 to about 3.25:1; a pH in a range of from about 6 to about 8;sodium ions at a concentration of at least 50 mmol/L; and apoly(ethylene oxide) surfactant selected from the group consisting of:(a) the poly(ethylene oxide) surfactant is represented by the structureof formula I:

wherein n is in the range of from about 9 to about 10; (b) thepoly(ethylene oxide) surfactant is represented by the structure offormula II:HO—(CH₂—CH₂—O—)₂₃—C₁₂H₂₅  Formula II; and (c) the poly(ethylene oxide)surfactant is represented by the structure of formula III:

wherein n is 100; contacting the magnesium sensing membrane with atleast one additional reagent selected from a wash reagent and a qualitycontrol reagent, wherein the at least one additional reagent comprisescalcium ions and magnesium ions present in a calcium:magnesium molarratio in a range of from about 1.5:1 to about 3.25:1; and wherein themagnesium sensing membrane is subjected to multiple calibrations withthe at least three separate calibration reagents and is stable for atleast four weeks.
 20. The method of claim 19, wherein the concentrationof the poly(ethylene oxide) surfactant present in each calibrationreagent is less than about 100 mg/L.